Fusion proteins and methods for stimulating plant growth, protecting plants from pathogens, and immobilizing Bacillus spores on plant roots

Abstract

The present invention is generally directed to fusion proteins containing a targeting sequence that targets the fusion protein to the exosporium of a Bacillus cereus family member. The invention also relates to recombinant Bacillus cereus family members expressing such fusion proteins, formulations containing the recombinant Bacillus cereus family members expressing the fusion proteins. Methods for stimulating plant growth and for protecting plants from pathogens by applying the recombinant Bacillus cereus family members or the formulations to plants or a plant growth medium are also described. The invention also relates to methods for immobilizing spores of a recombinant Bacillus cereus family member expressing a fusion protein on plant roots.

What is claimed is: 
     
       1. A method for stimulating plant growth comprising:
 applying a recombinant  Bacillus cereus  family member expressing a fusion protein to a plant seed, wherein applying the recombinant  Bacillus cereus  family member to the plant seed comprises: (a) applying the recombinant  Bacillus cereus  family member to the plant seed at the time of planting; or (b) coating the plant seed with a seed coating formulation comprising the recombinant  Bacillus cereus  family member and an agriculturally acceptable carrier; and 
 contacting the seed with a plant growth medium; 
 wherein the fusion protein comprises at least one plant growth stimulating protein or peptide and a targeting sequence or exosporium protein that targets the fusion protein to the exosporium of the recombinant  Bacillus cereus  family member, and wherein plants grown from plant seeds to which the recombinant  Bacillus cereus  family member has been applied exhibit increased growth as compared to plants grown from plant seeds to which the recombinant  Bacillus cereus  family member has not been applied, under the same conditions. 
 
     
     
       2. A method of  claim 1 , wherein the fusion protein comprises a targeting sequence comprising an amino acid sequence having at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 54%. 
     
     
       3. A method of  claim 2 , wherein the targeting sequence comprises an amino acid sequence having at least about 50% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 63%. 
     
     
       4. A method of  claim 2 , wherein the targeting sequence comprises an amino sequence having at least about 62% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 72%. 
     
     
       5. A method of  claim 2 , wherein the targeting sequence comprises an amino acid sequence having at least about 81% identity with amino acids 20-35 of SEQ ID NO: 1, wherein the identity with amino acids 25-35 is at least about 90%. 
     
     
       6. A method of  claim 2 , wherein the targeting sequence consists of:
 (a) a 16 amino acid sequence having at least about 43% identity with amino acids 20-35 of SEQ ID NO. 1, wherein the identity with amino acids 25-35 is at least about 54%; 
 (b) amino acids 1-35 of SEQ ID NO. 1; 
 (c) amino acids 20-35 of SEQ ID NO. 1; or 
 (d) SEQ ID NO. 1. 
 
     
     
       7. A method of  claim 1 , wherein the targeting sequence comprises the amino acid sequence GXT at its carboxy terminus, wherein X is any amino acid. 
     
     
       8. A method of  claim 1 , wherein the plant growth stimulating protein comprises an enzyme. 
     
     
       9. A method of  claim 8 , wherein the enzyme comprises a cellulase. 
     
     
       10. A method of  claim 9 , wherein the cellulase comprises an endocellulase, an exocellulase, or a β-glucosidase. 
     
     
       11. A method of  claim 10 , wherein the endocellulase comprises a  Bacillus subtilis  endoglucanase, a  Bacillus thuringiensis  endoglucanase, a  Bacillus cereus  endoglucanase, or a  Bacillus clausii  endoglucanase. 
     
     
       12. A method of  claim 8 , wherein the enzyme comprises a lipase. 
     
     
       13. A method of  claim 12 , wherein the lipase comprises a  Bacillus subtilis  lipase, a  Bacillus thuringiensis  lipase, a  Bacillus cereus  lipase, or a  Bacillus clausii  lipase. 
     
     
       14. A method of  claim 8 , wherein the enzyme comprises a lignin oxidase. 
     
     
       15. A method of  claim 14 , wherein the lignin oxidase comprises a lignin peroxidase, a laccase, a glyoxal oxidase, a ligninase, or a manganese peroxidase. 
     
     
       16. A method of  claim 8 , wherein the enzyme comprises a protease. 
     
     
       17. A method of  claim 16 , wherein the protease comprises a subtilisin, an acid protease, an alkaline protease, a proteinase, a peptidase, an endopeptidase, an exopeptidase, a thermolysin, a papain, a pepsin, a trypsin, a pronase, a carboxylase, a serine protease, a glutamic protease, an aspartate protease, a cysteine protease, a threonine protease, or a metalloprotease. 
     
     
       18. A method of  claim 8 , wherein the enzyme comprises a glycoside hydrolase. 
     
     
       19. A method of  claim 8 , wherein the enzyme comprises a phosphatase. 
     
     
       20. A method of  claim 19 , wherein the phosphatase comprises a phosphoric monoester hydrolase, a phosphomonoesterase, a phosphoric diester hydrolase, a phosphodiesterase, a triphosphoric monoester hydrolase, a phosphoryl anhydride hydrolase, a pyrophosphatase, a phytase, a trimetaphosphatase, or a triphosphatase. 
     
     
       21. A method of  claim 8 , wherein the enzyme comprises a nitrogenase. 
     
     
       22. A method of  claim 21 , wherein the nitrogenase comprises a Nif family nitrogenase. 
     
     
       23. A method of  claim 8 , wherein the enzyme comprises a nuclease. 
     
     
       24. A method of  claim 8 , wherein the plant growth medium is supplemented with a substrate for an enzyme. 
     
     
       25. A method of  claim 24 , wherein the substrate comprises tryptophan, an adenosine monophosphate, an adenosine diphosphate, an adenosine triphosphate, indole, a trimetaphosphate, ferrodoxin, acetoin, diacetyl, pyruvate, acetolactate, or a combination thereof. 
     
     
       26. A method of  claim 1 , comprising coating the plant seed with the seed coating formulation. 
     
     
       27. A method of  claim 1 , wherein the recombinant  Bacillus cereus  family member comprises  Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephensis , or a combination thereof. 
     
     
       28. A method of  claim 1 , wherein the plant growth stimulating protein or peptide comprises a peptide hormone. 
     
     
       29. A method of  claim 28 , wherein the peptide hormone comprises a phytosulfokine, clavata 3 (CLV3), systemin, ZmlGF, or a SCR/SP11. 
     
     
       30. A method of  claim 1 , wherein the plant growth stimulating protein or peptide comprises a non-hormone peptide. 
     
     
       31. A method of  claim 30 , wherein the non-hormone peptide comprises a RKN 16D10, Hg-Syv46, an eNOD40 peptide, RHPP, or kunitz trypsin inhibitor. 
     
     
       32. A method of  claim 1 , wherein the plant growth stimulating protein or peptide is physically attached to the exosporium of the recombinant  Bacillus cereus  family member. 
     
     
       33. A method of  claim 1 , further comprising inactivating the recombinant  Bacillus cereus  family member prior to application to the plant seed. 
     
     
       34. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-35 of SEQ ID NO: 1; 
 (b) a targeting sequence comprising amino acids 20-35 of SEQ ID NO: 1; 
 (c) a targeting sequence comprising SEQ ID NO: 1; or 
 (d) an exosporium protein comprising SEQ ID NO: 2. 
 
     
     
       35. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-27 of SEQ ID NO: 3; 
 (b) a targeting sequence comprising amino acids 12-27 of SEQ ID NO: 3; 
 (c) a targeting sequence comprising SEQ ID NO: 3; or 
 (d) an exosporium protein comprising SEQ ID NO: 4. 
 
     
     
       36. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-38 of SEQ ID NO: 5; 
 (b) a targeting sequence comprising amino acids 23-38 of SEQ ID NO: 5; 
 (c) a targeting sequence comprising SEQ ID NO: 5; or 
 (d) an exosporium protein comprising SEQ ID NO: 6. 
 
     
     
       37. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-28 of SEQ ID NO: 7; 
 (b) a targeting sequence comprising amino acids 13-28 of SEQ ID NO: 7; 
 (c) a targeting sequence comprising SEQ ID NO: 7; or 
 (d) an exosporium protein comprising SEQ ID NO: 8. 
 
     
     
       38. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-24 of SEQ ID NO: 9; 
 (b) a targeting sequence comprising amino acids 9-24 of SEQ ID NO: 9; 
 (c) a targeting sequence comprising SEQ ID NO: 9; or 
 (d) an exosporium protein comprising SEQ ID NO: 10. 
 
     
     
       39. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-33 of SEQ ID NO: 11; 
 (b) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 11; 
 (c) a targeting sequence comprising SEQ ID NO: 11; or 
 (c) an exosporium protein comprising SEQ ID NO: 12. 
 
     
     
       40. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-33 of SEQ ID NO: 13; 
 (b) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 13; 
 (c) a targeting sequence comprising amino SEQ ID NO: 13; or 
 (d) an exosporium protein comprising SEQ ID NO: 14. 
 
     
     
       41. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-43 of SEQ ID NO: 15; 
 (b) a targeting sequence comprising amino acids 28-43 of SEQ ID NO: 15; 
 (c) a targeting sequence comprising amino SEQ ID NO: 15; or 
 (d) an exosporium protein comprising SEQ ID NO: 16. 
 
     
     
       42. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-27 of SEQ ID NO: 17; 
 (b) a targeting sequence comprising amino acids 12-27 of SEQ ID NO: 17; 
 (c) a targeting sequence comprising amino SEQ ID NO: 17; or 
 (d) an exosporium protein comprising SEQ ID NO: 18. 
 
     
     
       43. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-33 of SEQ ID NO: 19; 
 (b) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 19; 
 (c) a targeting sequence comprising amino SEQ ID NO: 19; or 
 (d) an exosporium protein comprising SEQ ID NO: 20. 
 
     
     
       44. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-33 of SEQ ID NO: 21; 
 (b) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 21; 
 (c) a targeting sequence comprising amino SEQ ID NO: 21; or 
 (d) an exosporium protein comprising SEQ ID NO: 22. 
 
     
     
       45. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-24 of SEQ ID NO: 23; 
 (b) a targeting sequence comprising amino acids 9-24 of SEQ ID NO: 23; 
 (c) a targeting sequence comprising amino SEQ ID NO: 23; or 
 (d) an exosporium protein comprising SEQ ID NO: 24. 
 
     
     
       46. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-24 of SEQ ID NO: 25; 
 (b) a targeting sequence comprising amino acids 9-24 of SEQ ID NO: 25; 
 (c) a targeting sequence comprising amino SEQ ID NO: 25; or 
 (d) an exosporium protein comprising SEQ ID NO: 26. 
 
     
     
       47. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-30 of SEQ ID NO: 27; 
 (b) a targeting sequence comprising amino acids 15-30 of SEQ ID NO: 27; 
 (c) a targeting sequence comprising amino SEQ ID NO: 27; or 
 (d) an exosporium protein comprising SEQ ID NO: 28. 
 
     
     
       48. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-33 of SEQ ID NO: 29; 
 (b) a targeting sequence comprising amino acids 18-33 of SEQ ID NO: 29; 
 (c) a targeting sequence comprising amino SEQ ID NO: 29; or 
 (d) an exosporium protein comprising SEQ ID NO: 30. 
 
     
     
       49. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-24 of SEQ ID NO: 31; 
 (b) a targeting sequence comprising amino acids 9-24 of SEQ ID NO: 31; 
 (c) a targeting sequence comprising amino SEQ ID NO: 31; or 
 (d) an exosporium protein comprising SEQ ID NO: 32. 
 
     
     
       50. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-15 of SEQ ID NO: 33; 
 (b) a targeting sequence comprising amino SEQ ID NO: 33; or 
 (c) an exosporium protein comprising SEQ ID NO: 34. 
 
     
     
       51. A method of  claim 1 , wherein the fusion protein comprises:
 (a) a targeting sequence comprising amino acids 1-16 of SEQ ID NO: 35; 
 (b) a targeting sequence comprising amino SEQ ID NO: 35; or 
 (c) an exosporium protein comprising SEQ ID NO: 36.